A dipentene female black fly repellent, a preparation method and application thereof
By preparing dipentene solutions with concentrations ranging from 10⁻⁴ μg/μL to 10⁻¹ μg/μL, and utilizing gas chromatography-antennae potential coupling technology and behavioral experiments, female black-whiskered flies were repelled. This solved the problem of poor repellency in existing technologies and achieved the effect of reducing contact between female black-whiskered flies and the host, thus preventing vaginal myiasis.
Patent Information
- Application Number
- CN202311373336.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-20
- Publication Date
- 2026-11-06
- Estimated Expiration
- 2043-10-20
AI Technical Summary
Existing technologies are insufficient to effectively repel female black-whiskered flies, leading to increased opportunities for them to come into contact with the host, which in turn affects their mating and maggot-producing behavior, making it impossible to effectively prevent vaginal myiasis.
A repellent was prepared as a solution using dipentene at concentrations ranging from 10⁻⁴ μg/μL to 10⁻¹ μg/μL. Its effects on female black-bearded flies were determined using gas chromatography-antennae potential coupled with behavioral experiments, aiming to reduce their chances of contact with the host.
It effectively repels female black-whiskered flies, reduces their chances of contact with the host, significantly affects their mating and maggot-producing behavior, and prevents vaginal myiasis.
Smart Images
Figure CN117882715B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological control technology, specifically relating to a dipentene repellent for female black-bearded flies, its preparation method, and its application. Background Technology
[0002] The black-whiskered fly (Wohlfahrtia magnifica), also known as the flesh fly, was discovered and named by Schiner in 1862. It belongs to the genus *Wohlfahrtia brauer* of the subfamily Paramacronychiinae within the family Sarcophagidae of the order Diptera. The black-whiskered fly prefers warmth and light, and is primarily active in summer. It is a major pathogen causing bleeding wounds and myiasis in natural orifices in livestock (horses, camels, dogs, goats, sheep, cattle, etc.), and is also the sole pathogen causing vaginal myiasis in Bactrian camels. In addition, the larvae of the black-whiskered fly can cause various diseases of the ears, eyes, skin, and vulva in humans.
[0003] In my country, the black-bearded fly primarily causes vaginal myiasis in female camels, severely impacting their growth and development and hindering the camel industry, making it the leading cause of disease in female camels. Currently, myiasis control mainly relies on pesticides. While these pesticides kill the maggots in the parasitic parts, they are ineffective against black-bearded flies in the environment. Once the pesticide's effect wears off, the flies continue to produce maggots, leading to repeated infections. To change this passive, pesticide-based control approach, we need to explore proactive biological control measures. Summary of the Invention
[0004] The purpose of this invention is to solve the existing technical problems and provide a dipentene female black-whiskered fly repellent, its preparation method and application. This repellent can repel female black-whiskered fly adults to reduce their contact with the host, thereby affecting their mating and maggot-producing behavior, and providing a new approach for the treatment of vaginal myiasis in Bactrian camels.
[0005] This invention is achieved through the following technical solution:
[0006] A dipentene repellent for female black-bearded flies, the preparation method of which is as follows: dipentene and solvent are mixed to form a solution.
[0007] In the above technical solution, the solvent is n-hexane.
[0008] In the above technical solution, the concentration of dipentene is 10. -4 μg / μL to 10 -1 μg / μL.
[0009] In the above technical solution, the concentration of the dipentene is preferably 10. -4 μg / μL, 10 -3 μg / μL, 10 -2 μg / μL or 10 -1 μg / μL.
[0010] Application of a dipentene female black-bearded fly repellent in the prevention and control of livestock diseases: The above repellent is used to repel adult black-bearded flies, thereby reducing their contact with the host and affecting their mating and maggot-producing behavior.
[0011] In the above technical solution, the livestock are horses, camels, dogs, goats, sheep, cattle, etc.
[0012] In the above technical solution, the patient suffers from bleeding trauma and / or natural pore myiasis.
[0013] The beneficial effects of this invention are:
[0014] This invention discloses a dipentene repellent for female black-whiskered flies, its preparation method, and its application. In this invention, the antennal potential and behavioral response of the black-whiskered fly pheromone-dipentene were measured using gas chromatography-electroacoustic potential (GC-EAD), antennal potential (EAG), and behavioral experiments to understand the effect of dipentene on the behavior of black-whiskered flies. Experiments confirmed that using a concentration of 10... -4 μg / μL, 10 -3 μg / μL, 10 -2 μg / μL or 10 -1 A μg / μL dipentene blackfly repellent can repel female blackfly flies, thereby reducing the chance of contact between blackfly flies and Bactrian camels, and can effectively prevent vaginal myiasis caused by blackfly flies. Attached Figure Description
[0015] Figure 1 The image shows the GC-EAD reaction of dipentene by the black-whiskered fly. Note: The left image shows the GC-EAD reaction of dipentene by a female black-whiskered fly; the right image shows the GC-EAD reaction of dipentene by a male black-whiskered fly. In the images, FID represents the mass spectrum of the compound, EAD represents the antennal reaction of the black-whiskered fly to the compound, and the three EADs represent three replicate experiments of antennal potential.
[0016] Figure 2 The EAG reaction of black-whiskered flies to different concentrations of dipentene.
[0017] Figure 3 Comparison of the EAG reaction of black-bearded flies to dipentene.
[0018] Figure 4 The behavioral response of female black-whiskered flies to different concentrations of dipentene.
[0019] Figure 5 shows the behavioral responses of male Wohlfahrtia magnifica to different concentrations of dipentene.
[0020] Figure 6 is a comparison of the behavioral responses of Wohlfahrtia magnifica to dipentene.
[0021] Note: ns: no significant difference (P>0.05) *: significant difference (0.01<P<0.05) **: extremely significant difference (P<0.01).
[0022] For a person of ordinary skill in the art, other related drawings can be obtained from the above drawings without creative efforts. DETAILED DESCRIPTION
[0023] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solution of the present invention will be further described below in combination with specific examples.
[0024] Example 1: GC-EAD responses of Wohlfahrtia magnifica to dipentene
[0025] (1) Sample preparation: The purchased dipentene (≥98%) was prepared to 10 with HPLC-grade n-hexane -2 μg / μL test sample, and stored in a -20°C refrigerator for later use.
[0026] (2) Capillary preparation: Soften the capillary with an alcohol lamp, and manually pull the capillary until the tip is just large enough to hold the insect antenna, keeping the process as smooth and stable as possible. After the capillary is prepared, fill it with a conductive liquid, and 0.9% normal saline is filled in this experiment.
[0027] (3) Antenna connection:
[0028] In this experiment, female and male Wohlfahrtia magnifica of about 7 days old were selected to test dipentene at a concentration of 10 -2 μg / μL. Each sample was tested in triplicate for female and male Wohlfahrtia magnifica, and each antenna was used only once. Under a microscope, the antenna of Wohlfahrtia magnifica was cut off from the base, and both ends of the antenna were connected to the two glass electrodes of the electroantennograph using glass capillaries containing normal saline. The base of the antenna was connected to the reference electrode, and the top was connected to the recording electrode. After the EAD baseline of the electroantennograph was stabilized, sample injection was started, and the signal recorded by the recording electrode was amplified and converted by the Syntech IDAC-2 signal collector, and then data acquisition and analysis were performed through GC-EAD software.
[0029] (4) Gas chromatography (GC) conditions:
[0030] The initial temperature was 50℃ and held for 1 min. The temperature was then increased to 120℃ at a rate of 5℃ / min and held for 1 min. The temperature was then increased to 150℃ at a rate of 10℃ / min and held for 1 min. Finally, the temperature was increased to 280℃ at a rate of 20℃ / min and held for 10 min. The injection volume was 1 μL. The injection was splitless.
[0031] Example 2: EAG reaction of black-bearded flies to dipentene
[0032] (1) Sample preparation: Dipentene was prepared into a solution of 10 μL using chromatographic grade n-hexane. -4 μg / μL, 10 -3 μg / μL, 10 -2 μg / μL and 10 -1 The test sample of μg / μL should be stored at -20℃ for later use.
[0033] (2) Check if the level of distilled water in the gas washing bottle is half full, and add more if necessary.
[0034] (3) Turn on the power supply of the CS-55 stimulation airflow generator and the relevant software on the computer.
[0035] (4) Prepare the antennae of female and male black-bearded flies that are about 7 days old and attach the antennae.
[0036] (5) Drop 10 μL of the sample to be tested evenly onto the filter paper strip, place the filter paper strip into the blue gun tip, and stimulate it after the solvent evaporates for 5 minutes. Use n-hexane as a blank control. Perform EAG determination of the blank control after each antennae is tested, thereby correcting the EAG reaction value of the sample.
[0037] (6) Stimulation conditions: stimulation time is 0.2s, stimulation interval is 60s, stimulation airflow rate is 300mL / min, and continuous airflow is set to 1000mL / min.
[0038] (7) Each sample was measured three times to distinguish between male and female.
[0039] (8) The EAG measurement results are recorded by the EAG signal recording and analysis program. The calculation method for the relative response value of the sample antennal potential (EAG relative response value) is as follows:
[0040] EAG relative response value = (2 × antennal potential response value of the test sample) ÷ (antennal potential response value of the solvent control before the test sample + antennal potential response value of the solvent control after the test sample)
[0041] (9) Experimental results: One-way ANOVA and t-test were performed on the data using GraghPad Prism 8.0.1 and plotted.
[0042] Example 3: Behavioral response of black-bearded flies to dipentene
[0043] (1) Connect the following devices sequentially using a water purifier tube: atmospheric sampler (outlet) → drying tower containing activated carbon → humidification device → odor bottle → Y-shaped test tube. Maintain the airflow in both arms at 1 L / min. Before the experiment, all instruments should be cleaned three times each with distilled water and alcohol to reduce errors. Select black-bearded flies around 5 days old for behavioral testing. Place only one black-bearded fly at the straight arm of the Y-shaped test tube each time, with a reaction time of 5 min. Record the result when the fly enters more than 1 / 2 of the side arm and stays for at least 30 seconds. If no selection is made within 5 min, stop the behavioral test for that fly and do not record it. Use 20 female and 20 male black-bearded flies in each group, repeat the test 3 times, and test each fly only once at the same concentration. Use distilled water as a control. The experiment is conducted at room temperature.
[0044] (2) The experimental data were analyzed and plotted using the GraghPad Prism test. If the response of black-bearded flies to the compound was significantly higher than that to the control group, then the compound had an attractive effect on black-bearded flies; conversely, if the response of black-bearded flies to the control group was significantly higher than that to the compound, then the compound had a repellent effect on black-bearded flies; if the responses of black-bearded flies to both were not significant, then it could not be determined whether the compound had an attractive or repellent effect on black-bearded flies.
[0045] Table 1. Relative EAG response values of black-bearded flies to different concentrations of dipentene.
[0046]
[0047]
[0048] Results Analysis
[0049] Depend on Figure 1 It can be seen that adult male and female black-bearded flies of different ages affect 10 -2 Dipentene at μg / μL all exhibited the GC-EAD reaction.
[0050] The results of EAG assays on female black-bearded flies with different concentrations of dipentene showed that female black-bearded flies produced antennal responses to different concentrations of dipentene, and the relative EAG response value increased with increasing dipentene concentration, although there was no significant difference in the relative EAG response value (P>0.05) (Table 1). Figure 2 (Left image).
[0051] The results of EAG assays on male black-whiskered flies to different concentrations of dipentene showed that male black-whiskered flies produced antennal responses to all concentrations of dipentene, and the relative EAG response value increased with increasing dipentene concentration, although there was no significant difference in the relative EAG response value (P > 0.05) (Table 1). Figure 2 (Right image).
[0052] The results of comparing the EAG responses of female and male black-bearded flies to dipentene showed no significant difference between the two groups (P > 0.05). Figure 3 ).
[0053] Experimental results show that 10 -3 μg / μL and 10 -1 Dipentene at a concentration of μg / μL exhibited a highly significant repellent effect against female black-bearded flies (P < 0.01); a concentration of 10 μg / μL... -2 It exhibits significant repellency at a concentration of μg / μL (0.01 < P < 0.05); when the dipentene concentration is 10 μg / μL... -4 At μg / μL, it had no significant attraction or repulsion effect on female black-bearded flies (P>0.05). Figure 4 ).
[0054] Experimental results show 10 -4 μg / μL, 10 -3 μg / μL, 10 -2 μg / μL and 10 -1 Four concentrations of dipentene (μg / μL) showed no significant behavioral response in male black-bearded flies (P > 0.05). Figure 5 ).
[0055] Behavioral responses of different sexes of black-bearded flies to different concentrations of dipentene, as follows: Figure 6 As shown in the analysis results, male black-bearded flies are effective against 10... -3 μg / μL and 10 -1 The behavioral response to dipentene at μg / μL was significantly higher in females than in females (0.01 < P < 0.05); 10 -4 μg / μL and 10 -2 Dipentene at μg / μL did not elicit a significant behavioral response in either female or male black-bearded flies (P > 0.05).
[0056] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.
Claims
1. The use of a dipentene female black-whiskered fly repellent in the preparation of drugs for bleeding wounds or natural canal fly myiasis, characterized in that, The repellent is prepared by formulating dipentene into a solution with a solvent, the concentration of the dipentene being 10 -4 μg / μL to 10 -1 μg / μL.
2. The application according to claim 1, characterized in that: The solvent is n-hexane.
3. The application according to claim 1, characterized in that: The concentration of the dipentene is 10. -4 μg / μL, 10 -3 μg / μL, 10 -2 μg / μL or 10 -1 μg / μL.